Study on the Degradation of Glycosaminoglycans by Canine Liver Lysosomal Enzymes

Abstract

Hyaluronic acid and its oligosaccharides (tetra-, hexa-, and octasaccharides) were digested by canine liver lysosomes at pH 4.0. The degradation products were separated by gel filtration and ion exchange chromatography. The contribution of hyaluronidase and exoglycosidases (β-glucuronidase and β-N-acetylhexosaminidase) on the degradation of hyaluronic acid was inferred from the distribution of the oligosaccharides, glucuronic acid, and N-acetylglucosamine in the digestion mixture. Hyaluronic acid was degraded into a large amount of even-numbered oligosaccharides and to a trace amount of free glucuronic acid in the initial stage of the digestion. On the other hand, the prolonged digestion study showed an increase of odd-numbered oligosaccharides such as the tri- and pentasaccharides in proportion to an increase of free glucuronic acid. On the degradation of the hyaluronic acid oligosaccharides, tetra- and hexasaccharides gave tri- and pentasaccharides as the main oligosaccharides in the degradation products respectively. Octasaccharide was not degraded into heptasaccharide by the action of β-glucuronidase but into a mixture of di-, tetra-, and hexasaccharides by hyaluronidase. The activity of β-glucuronidase for tetra- and hexasaccharides was higher than that of β-N-acetylhexosaminidase for tri- and pentasaccharides. On the basis of these findings, hyaluronic acid is first depolymerized by hyaluronidase alone and octasaccharide is the minimum substrate size in the degradation by hyaluronidase. Hexasaccharide and other smaller oligosaccharides are degraded by the action of exoglycosidases. The level of β-N-acetylhexosaminidase activity is a limiting factor in the degradation by the concerted action of exoglycosidases.